Accessibility / Report Error

The existence of only one haplotype of Leishmania major in the main and potential reservoir hosts of zoonotic cutaneous leishmaniasis using different molecular markers in a focal area in Iran

Abstract

Introduction

Leishmania major is the causative agent of zoonotic cutaneous leishmaniasis (ZCL), and great gerbils are the main reservoir hosts in Iran. Abarkouh in central Iran is an emerging focal point for which the reservoir hosts of ZCL are unclear. This research project was designed to detect any Leishmania parasites in different wild rodent species.

Methods

All rodents captured in 2011 and 2012 from Abarkouh district were identified based on morphological characteristics and by amplification of the rodent cytochrome b (Cyt b) gene. To detect Leishmania infection in rodents, deoxyribonucleic acid (DNA) of each ear was extracted. Internal transcribed spacer-ribosomal deoxyribonucleic acid (ITS-rDNA), microsatellites, kinetoplast deoxyribonucleic acid (kDNA) and cytochrome b genes of Leishmania parasites were amplified by polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) and sequencing were employed to confirm the Leishmania identification.

Results

Of 68 captured rodents in the region, 55 Rhombomys opimus were identified and nine Leishmaniainfections (9/55) were found. In addition, eight Meriones libycus and two Tatera indicawere sampled, and one of each was confirmed to be infected. Two Meriones persicus and one Mus musculuswere sampled with no infection.

Conclusions

The results showed that all 11 unambiguously positive Leishmania infections were Leishmania major. Only one haplotype of L. major(GenBank access No. EF413075) was found and at least three rodents R. opimus, M. libycus and T. indica—appear to be the main and potential reservoir hosts in this ZCL focus. The reservoir hosts are variable and versatile in small ZCL focal locations.

Leishmania parasite; Zoonotic cutaneous leishmaniasis; Rodents; Haplotype; Iran


INTRODUCTION

Leishmaniasis is one of the nine emerging individual infectious diseases that have been largely neglected around the world and in the Middle East11. World Health Organization Control of Leishmaniasis. Technical report series 949 of WHO Expert Committee. Geneva: WHO; 2010..

In Iran, leishmaniasis is observed in three clinical forms: zoonotic cutaneous leishmaniasis (ZCL), anthroponotic cutaneous leishmaniasis (ACL) and zoonotic visceral leishmaniasis (ZVL). ZCL caused by Leishmania major has a health as well as socioeconomic impacts in Iran. ZCL has been reported in rural regions of Iran in 15 of 31 Provinces,including: Bushehr, Hormozgan and Fars in the south22. Zare S, Baghestani S. Cutaneous Leishmaniasis in Hormozgan, Iran. Int J Dermatol 2001; 40:29-631.,33. Maraghi S, Samarbaf Zadeh A, Sarlak AA, Ghasemian M, Vazirianzadeh B. Identification of Cutaneous Leishmaniasis Agents by Nested Polymerase Chain Reaction (Nested-PCR) in Shush City, Khuzestan Province, Iran. Iran J Parasitol 2007; 2:13-15.; Ilam and Khuzestan in the southwest and west44. Jahani MR, Gharavi MJ, Hadi Shirzad H. Passive Detection of Cutaneous Leishmaniasis in Police Personnel Deployed in the Provinces of Isfahan, Ilam, Bushehr, Khorasan and Khuzestan, Iran. Iran J Public Health 2003; 32:23-27.,55. Sharbatkhori M, Spotin A, Taherkhani H, Roshanghalb M, Parvizi P. Molecular variation in Leishmania parasites from sandflies species of a zoonotic cutaneous leishmaniasis in northeast of Iran. J Vector Borne Dis 2013; 51:16-21.; Golestan, Khorasan and Semnan in the northeast and north44. Jahani MR, Gharavi MJ, Hadi Shirzad H. Passive Detection of Cutaneous Leishmaniasis in Police Personnel Deployed in the Provinces of Isfahan, Ilam, Bushehr, Khorasan and Khuzestan, Iran. Iran J Public Health 2003; 32:23-27.66. Alavinia SM, Arzamani K, Reihani MH, Jafari J. Some epidemiological aspects of cutaneous leishmaniasis in Northern Khorasan Province, Iran. Iran J Arthropod Borne Dis 2009; 3:50-54.; and Isfahan in the central region77. Parvizi P, Ready PD. Nested PCRs and sequencing of nuclear ITS rDNA fragments detect three Leishmania species of gerbils in sandflies from Iranian foci of zoonotic cutaneous leishmaniasis. TM & IH 2008; 13:1159-1171..

During the life cycle of ZCL, which depends on the geographical location of the disease, sandflies act as vectors of Leishmania, Bartonella bacilliformis and some arboviruses, and wild rodents are considered to be the reservoir hosts88. Ready PD. Biology of Phlebotomine sand flies as vectors of disease agents. Ann Rev Entomol 2013; 58:227-250.. Many investigations have been conducted on different aspects of ZCL in naturally important foci in Iran99. Parvizi P, Bordbar A, Najafzadeh N. Detection of Wolbachia pipientis, including a new strain containing the wsp gene, in two sister species of Paraphlebotomus sandflies, potential vectors of zoonotic cutaneous leishmaniasis. Mem Inst Oswaldo Cruz 2013; 108:414-420.1111. Mirzaei A, Rouhani S, Kazerooni PA, Farahmand M, Parvizi P. Molecular detection and conventional identification of Leishmania species in reservoir hosts of zoonotic cutaneous leishmaniasis in Fars province, South of Iran. Iran J Parasitol 2013; 8:280-288., although some areas of neighboring Provinces have been neglected for unknown reasons. Predisposing factors, such as increasing migration of patients from endemic foci to potential areas, irregular construction and urbanization and changing sandfly fauna in the region affect the distribution and survival of ZCL99. Parvizi P, Bordbar A, Najafzadeh N. Detection of Wolbachia pipientis, including a new strain containing the wsp gene, in two sister species of Paraphlebotomus sandflies, potential vectors of zoonotic cutaneous leishmaniasis. Mem Inst Oswaldo Cruz 2013; 108:414-420.,1212. Ready PD. Leishmaniasis emergence and climate change. Rev Sci Tech 2008; 27:399-412..

Yazd Province in central Iran is one of these regions, and the number of cases of ZCL has been increasing since 19811010. Jafari R, Najafzadeh N, Sedaghat MM, Parvizi P. Molecular characterization of sandflies and Leishmania detection in main vector of zoonotic cutaneous leishmaniasis in Abarkouh district of Yazd province, Iran. Asian Pac J Trop Med 2013; 6:792-797.. The official reports from the health center in Yazd Province has demonstrated that the number of cases of CL in the Ardakan area (which is an important potential focus in southwestern Yazd) increased from 1996 to 1997 (total: 372 cases), which may lead to monitoring and surveillance activities in this district1313. Safari Z, Saadati M, Doroudian M, Hossaini SM, Hashemi M, Rezai zarchi S. Cutaneous leishmaniasis characterization in the Center Part of Iran; is that an emerging disease? Adv Environ Biol 2011; 5:3464-3470.. A few studies have examined some cities in Yazd Province (Ardakan, Taft, Bafgh, and Khatam cities); however, no molecular-epidemiologic investigations have been performed in Abarkouh district yet. Abarkouh has many historical places (such as Abarkouh's Cedar) and tourism values therefore, tourists play an important role in spreading the infection to other Provinces. Thus, the isolation, detection and identification of Leishmania spp. in rodents are essential for disease prognosis, diagnosis methods, the monitoring of clinical outcomes, epidemiological perspectives and treatment program planning. Some reports have demonstrated that Rhombomys opimus and Meriones libycus are the most important reservoir hosts of ZCL in the neighboring Provinces of Isfahan and Shiraz1414. Akhavan AA, Yaghoobi-Ershadi MR, Mirhendi H, Alimohammadian MH, Rassi Y, Shareghi N, Jafari R, Arandian MH, Abdoli H, Ghanei M, Jalali-zand N, Khamesipour A. Molecular epizootiology of Rodent leishmaniasis in hyperendemic area of Iran. Iran J Public Health 2010; 39:1-7.,1515. Mirzaei A, Rouhani S, Taherkhani H, Farahmand M, Kazemi B, Hedayati M, et al. Isolation and detection of Leishmania species among naturally infected Rhombomys opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkemen Sahara, North East of Iran. Exp Parasitol 2011; 129:375-380.. The geographical distribution of reservoir hosts of ZCL in different regions of Iran is shown in Figure 11111. Mirzaei A, Rouhani S, Kazerooni PA, Farahmand M, Parvizi P. Molecular detection and conventional identification of Leishmania species in reservoir hosts of zoonotic cutaneous leishmaniasis in Fars province, South of Iran. Iran J Parasitol 2013; 8:280-288..

FIGURE 1
Map of Abarkouh showing sampling regions and the geographical distribution of reservoir hosts of ZCL in different regions of Iran. L: Leishmania; ZCL: zoonotic cutaneous leishmaniasis.

For population genetic studies and species identification, we utilized the cytochrome b gene of rodents, cytochrome b(Cyt b) of Leishmania, ITS1-5.8S ITS2 ribosomal deoxyribonucleic acid (rDNA), kinetoplast deoxyribonucleic acid (kDNA) and microsatellite deoxyribonucleic acid (DNA) genes were employed for detection of any Leishmania infection. Low intracellular polymorphism and readable sequences are important advantages of internal transcribed spacer-ribosomal deoxyribonucleic acid (ITS-rDNA) in molecular identifications77. Parvizi P, Ready PD. Nested PCRs and sequencing of nuclear ITS rDNA fragments detect three Leishmania species of gerbils in sandflies from Iranian foci of zoonotic cutaneous leishmaniasis. TM & IH 2008; 13:1159-1171.,1616. Parvizi P, Mauricio I, Aransay AM, Miles MA, Ready PD. First detection of Leishmania major in peridomestic Iranian sandflies: comparison of nested PCR of nuclear ITS ribosomal DNA and semi-nested PCR of minicircle kinetoplast DNA. Acta Trop 2005; 93:75-83.1818. Maleki-Ravasan N, Oshaghi MA, Javadian E, Rassi Y, Sadraei J, Mohtarami F. Blood meal identification in field-captured sandflies: comparison of PCR RFLP and ELISA assays. Iran J Arthropod Borne Dis 2009; 3:8-18.. The kinetoplast in Trypanosomatidae contains nearly 10,000 small circular DNAs (kDNA minicircle). This minicircle comprises a variable region (600bp) and a conserved region (120bp). Microsatellite markers in Leishmania parasites are co-dominant and allelic and combine 1-7 nucleotide units into short, tandemly repeated DNA sequences1919. Oryan A, Shirian S, Tabandeh MR, Hatam GR, Randau G, Daneshbod Y. Genetic diversity of Leishmania major strains isolated from different clinical forms of cutaneous leishmaniasis in southern Iran based on minicircle kDNA. Infect Genet Evol 2013; 19:226-31.,2020. Jamjoom MB, Ashford RW, Bates PA, Kemp SJ, Noyes HA. Polymorphic microsatellite repeats are not conserved between Leishmania donovani and Leishmania major. Mol Ecol Notes 2002; 2:104-106.. Currently, multilocus microsatellite typing (MLMT) is being used widely in population genetic studies in different species of Leishmania parasites2121. Kuhls K, Chicharro C, Canavate C, Cortes S, Campino L, Haralambous C, et al. Differentiation, Gene Flow, among European populations of Leishmania infantum MON-1. PLoS Negl Trop Dis 2008; 2:261.,2222. Russell R, Iribar MP, Lambson B, Brewster S, Blackwell JM, Dye C, et al. Intra and inter-specific microsatellite variation in the Leishmania subgenus Viannia. Mol Biochem Parasit 1999; 103:71-77.. Minicircle kDNA and microsatellite ITS-rDNA are also well known as molecular markers for the detection of Leishmaniainfections77. Parvizi P, Ready PD. Nested PCRs and sequencing of nuclear ITS rDNA fragments detect three Leishmania species of gerbils in sandflies from Iranian foci of zoonotic cutaneous leishmaniasis. TM & IH 2008; 13:1159-1171.,2121. Kuhls K, Chicharro C, Canavate C, Cortes S, Campino L, Haralambous C, et al. Differentiation, Gene Flow, among European populations of Leishmania infantum MON-1. PLoS Negl Trop Dis 2008; 2:261..

The Cyt b gene encodes the central catalytic subunit of an enzyme present in the respiratory chain of mitochondria and exists in almost all organisms. This gene has been broadly used for phylogenetic studies and identification of animals and plants2323. Degli esposti M, De Vries S, Crimi M, Ghelly A, Patarnello T, Meyer A. Mitochondrial cytochrome b: evolution and structure of the protein. Biochim Biophys Acta 1993; 1143:243-271.. The Cyt b gene of the genus Leishmania consists of two regions: the edited region (the most 5′ region of 23bp), which undergoes ribonucleic acid (RNA) editing, and the non-edited region (the 3′ region of 1,056bp)2323. Degli esposti M, De Vries S, Crimi M, Ghelly A, Patarnello T, Meyer A. Mitochondrial cytochrome b: evolution and structure of the protein. Biochim Biophys Acta 1993; 1143:243-271..

Since the rodent fauna, the Leishmania species and their infection rate in Abarkouh district of Yazd Province, Iran, haves not been elucidated completely; we have designed this study in order to investigate these aspects of ZCL.

METHODS

Study area, sampling and laboratory methods

This cross-sectional/descriptive study was performed in 2011 and 2012, and rodent samples were obtained from 5 villages across Abarkouh district, Yazd Province, central Iran, including: Abarghasr, Haroni, Khorram abad, Gonabad and Chahgir.

Abarkouh district is situated between Fars (southern Iran) and Isfahan (central Iran) Provinces (Figure 1). These Provinces are considered hyper-endemic regions that are important sites for ZCL77. Parvizi P, Ready PD. Nested PCRs and sequencing of nuclear ITS rDNA fragments detect three Leishmania species of gerbils in sandflies from Iranian foci of zoonotic cutaneous leishmaniasis. TM & IH 2008; 13:1159-1171.. Abarkouh district, with an altitude of 1,510 meters above sea level (a.s.l.) (4,954 feet), geographic coordinates of ″31°07′44″N 53°16′57″E31.13°N 53.28°E″ (Figure 1) and a population of approximately 21,818 people, is located in Yazd Province in central Iran. Due to its hot and dry climate and its proximity to Isfahan and Fars Provinces, Abarkouh is considered a new and emerging focus of ZCL as well.

The rodents were sampled in Abarkouh area 120km southwest of Yazd Province, using wooden and wire traps. To identify active colonies of rodents within a diameter of 1-1.5km around villages in Abarkouh, approximately 30-40 live traps were used, and the rodents were captured monthly by baiting with dates and cucumbers. The genus and species of each rodent were determined based on external features, including: ears, color, tail, body measurements, teeth, feet and cranium1515. Mirzaei A, Rouhani S, Taherkhani H, Farahmand M, Kazemi B, Hedayati M, et al. Isolation and detection of Leishmania species among naturally infected Rhombomys opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkemen Sahara, North East of Iran. Exp Parasitol 2011; 129:375-380.,2424. Ziaei H. A field guide for identifying of Iranian desert mammalians. 1st ed. Tehran: Iranian Environment Organization; 1996.,2525. Parvizi P, Moradi G, Akbari G, Ready PD, Farahmand M, Piazak N, et al. PCR Detection and sequencing of parasite ITS rDNA gene from reservoirs host of zoonotic cutaneous leishmaniasis in central of Iran. Parasitol Res 2008; 103:1273-1278..

Each protocol and method applied in this survey was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the Human and Animal Research Ethics Committee of the Pasteur Institute of Iran.

Two impression touch slides were obtained from both ears of each rodent by scratching. For brief microscopic observation, rodent samples were collected from the ears after removing the hair and making small scratches from which to extract serous fluid, which was then fixed on a microscopic slide with methanol and stained for 30min with Giemsa diluted 1:10. The slides were then observed under a light microscope to detect the presence of Leishman bodies.

Furthermore, serous fluid from the rodents' ears was injected into Balb/C mice to monitor for the appearance of Leishmania infection lesions. Prepared serous fluid from infected Balb/C mice accompanied by serous fluid from scratches from each ear of rodents was cultured in Novy-MacNeal-Nicolle (NNN) medium. Subsequently, the cultures were incubated at 22°C for 6 weeks. The cultures were checked at two-day intervals until they reached the growth phase (log phase) based on observation using an inverted microscope. Positive cultures were confirmed by the presence of promastigotes, which were sub-cultured into restriction fragment length polymorphism (RFLP) medium weekly.

The harvested promastigotes from the early stationary phase (approximately 2×106 promastigotes/ml) and serous fluid from each ear of the rodent were injected subcutaneously into the base tail of a Balb/C mouse. Inoculated Balb/C mice were examined weekly for the appearance of lesions at the injection site for 6 months. Samples from infected Balb/C mice with cutaneous lesions were used for DNA extraction.

Molecular methods

Smears prepared from infected Balb/C mice, with serous fluid and/or cuts from each rodent ear, were kept in separate 1.5ml microtubes containing 100µl phosphate-buffered saline (PBS) and then centrifuged briefly three times at 13,000rpm. The PBS was discarded. Each rodent ear was placed in a 1.5ml microtube and placed in liquid nitrogen 3 times for 3min each. The genomic DNA of each rodent and any parasite within was extracted using the ISH-Horovize method and a GeNet BIO kit (Global Gene Network South Korea); these procedures were carried out in the systematic molecular laboratory of the Pasteur Institute in a room where amplified and cloned DNAs were never processed1111. Mirzaei A, Rouhani S, Kazerooni PA, Farahmand M, Parvizi P. Molecular detection and conventional identification of Leishmania species in reservoir hosts of zoonotic cutaneous leishmaniasis in Fars province, South of Iran. Iran J Parasitol 2013; 8:280-288.,2525. Parvizi P, Moradi G, Akbari G, Ready PD, Farahmand M, Piazak N, et al. PCR Detection and sequencing of parasite ITS rDNA gene from reservoirs host of zoonotic cutaneous leishmaniasis in central of Iran. Parasitol Res 2008; 103:1273-1278..

The DNA samples extracted from rodent tissues were used in polymerase chain reaction (PCR) to amplify a 624bp fragment of the cytochrome b gene (Cyt b) from the mitochondrial DNA to accurately identify the rodent species. We followed the protocol of Kent and Norris (Table 1)2626. Kent R, Norris DE. Identification of mammalian blood meals in mosquitoes by a multiplex polymerase chain reaction targeting cytochrome b. Am J Trop Med Hyg 2005; 73:336-342..

TABLE 1
- Primer sequences and conditions used for all employed genes for the identification of Leishmania parasites within rodents.

The internal transcribed spacer-ribosomal deoxyribonucleic acid gene was amplified for the detection of Leishmania infection using ITS1-5.8SrRNA-ITS2 fragments, with ITS1F as the forward primer and ITS2R4 as the reverse primer (Table 1)77. Parvizi P, Ready PD. Nested PCRs and sequencing of nuclear ITS rDNA fragments detect three Leishmania species of gerbils in sandflies from Iranian foci of zoonotic cutaneous leishmaniasis. TM & IH 2008; 13:1159-1171..

To perform RFLP analysis, the PCR products were blunt digested using endonuclease BsuR1 (HaeIII) (Fermentas, Life Sciences, Germany) in the recognition site pattern GG↓CC, as recommended by the manufacturer. Enzyme selection was performed by analyzing sequences of Leishmania reference species with CLC DNA Workbench 5.2 software (CLC bio A/s, Aarhus, Denmark)2727. Bordbar A, Parvizi P. High infection frequency, low diversity of Leishmania major and first detection of Leishmania turanica in human in northern Iran. Acta Trop 2014; 133:69-72..

The primer sets LINR4 (forward), LIN17 (first-step reverse) and LIN19 (second-step reverse) were used in the semi-nested PCR for the minicircle kDNA gene1616. Parvizi P, Mauricio I, Aransay AM, Miles MA, Ready PD. First detection of Leishmania major in peridomestic Iranian sandflies: comparison of nested PCR of nuclear ITS ribosomal DNA and semi-nested PCR of minicircle kinetoplast DNA. Acta Trop 2005; 93:75-83.. The primers anneal within the conserved area of the minicircle and are based on the conserved sequence blocks recognized by Brewster and Baker (Table 1)2828. Brewster S, Barker DC. Analysis of minicircle classes in Leishmania (Viannia) species. T Roy Soc Trop Med H 2002; 96 (suppl I):55-63..

The third method used for Leishmania infection identification was microsatellite ITS-rDNA analysis; the protocol used in this assay was designed by Parvizi et al.2525. Parvizi P, Moradi G, Akbari G, Ready PD, Farahmand M, Piazak N, et al. PCR Detection and sequencing of parasite ITS rDNA gene from reservoirs host of zoonotic cutaneous leishmaniasis in central of Iran. Parasitol Res 2008; 103:1273-1278.. The primers were ITSMF1 (forward) and ITSMR2 (reverse) (Table 1).

To detect Leishmania infection, we also used a fragment of the cytochrome b gene from mitochondrial DNA, and the primers used in this amplification were LCBF1 (forward) and LCBR2 (reverse) (Table 1)1717. Luyo-Acero GE, Uezato H, Oshiro M, Takei K, Kariya K, Katakura K, et al. Sequence variation of the Cytochrome b gene of various human infecting members of the genus Leishmania and their phylogeny. Parasitol 2004; 128:483-491..

After amplification, the DNA samples were excised, purified and sequenced using an ABI PRISM TM310 automated sequencer (Applied Biosystems, USA). The sequences obtained were edited and aligned with database sequences using SequencherTM v. 4.4 software to identify unique sequences (= haplotypes), which were analyzed phylogenetically using MEGA5.05 software2929. Parvizi P, Alaeenovin E, Kazerooni PA, Ready PD. Low diversity of Leishmania parasites in sandflies and the absence of the great gerbil in foci of zoonotic cutaneous leishmaniasisin Fars province, southern Iran. T Roy Soc Trop Med H 2013; 107:356-362.,3030. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, et al. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011; 10:2731-2739..

Ethical considerations

This study was approved by the Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.

RESULTS

In total, 68 rodents were captured in five villages in Abarkouh district (Figure 1; Table 2). Thirty-three of 68 rodents were alive and transported to the Pasteur Institute of Iran, Tehran, for further studies using conventional and molecular methods. Thirty-five rodents were dead after being caught by wire and wooden live traps at sampling sites. The ears of these dead rodents were used only for molecular methods. Based on morphological characteristics and rodent molecular markers (Cyt bsequences), five species were identified. The most abundant rodent was R. opimus (55/68: 81%). The frequencies and abundances of the other rodent species were as follows: M. libycus(8/68: 12%), Meriones persicus (2/68: 3%), Tatera indica (2/68: 3%) and Mus musculus (1/68: 1%), respectively (Table 2).

TABLE 2
Leishmania infections among different rodents captured in Abarkouh district in Yazd Province, Iran, using conventional and molecular methods.

Eleven of 68 (16%) rodents were found to be infected with Leishmania parasites using molecular methods (3/68 (4.4%) using ITS-rDNA, 10/68 (14%) using microsatellites, 3/68 (4.4%) using minicircle kDNA and 4/68 (5.8%) by amplifying Cyt b from Leishmania parasites). At least 3 of 5 rodent species had Leishmania infections (Figure 2). Only two of 33 (6.06%) live rodents were found to be Leishmaniapositive using conventional methods, including impression touch smears from the ear, light microscope observation, culturing in NNN and inoculating in Balb/C mice (Table 2).

FIGURE 2
A: Electrophoresis image of Cyt b gene amplification in Leishmania infection among different rodent species of Abarkouh district, Yazd Province, Iran. B: RFLP of ITS-rDNA gene electrophoresis image after digestion with BsuR1 (HaeIII) enzyme of PCR products using In-Silico software (CLC bio A/s, Aarhus, Denmark) of Leishmania infection among different rodent species of Abarkouh district, Yazd Province, Iran (Bordbar and Parvizi 2013) (+Ve contains Leishmania major parasite PCR product without the enzyme effect, and +Ve (enz) is a Leishmania major parasite PCR product with the enzyme effect). Cyt b: cytochrome b; RFLP: Restriction fragment length polymorphism; ITS-rDNA: internal transcribed spacer-ribosomal deoxyribonucleic acid; PCR:polymerase chain reaction. +ve: positive sample, enz: with enzyme.

The most interesting result was that despite the low number of captured rodents, five different rodent species were collected and identified. Leishmania infection was detected from three of these species, and for the first time, T. indica was captured in Abarkouh district and identified both morphologically through diagnostic keys and molecularly by sequencing of the Cyt b gene. In addition, one of the two T. indica specimens was infected with L. major.

To find identify additional Leishmania parasite infections and molecular variation rates among collected samples, different genes were employed. Standard and semi-nested PCR were used to amplify ITS-rDNA, microsatellites, kDNA and Cyt b genes from Leishmania parasites (Figure 2).

All 11 Leishmania-positive samples were analyzed using RFLP and sequencing to definitively identify Leishmania species (Figure 2). With RFLP, which allows for the differentiation of each species unambiguously, two fragments of 120 and 310bp belonging to L. major were obtained (Figure 2).

All sequences from positive samples by ITS-rDNA gene were blasted and confirmed to be most similar to L. major, and only L. major with one common haplotype (GenBank accession No. EF413075) was found after direct sequencing, editing, aligning and comparing with the sequences submitted to GenBank using Sequencher TM v. 4.4 and phylogenetic analysis by MEGA5.05 software.

DISCUSSION

In our study, only the L. major parasite with one common haplotype (GenBank access No. EF413075) was firmly identified in three rodent species. Leishmania parasites have been isolated from all three species in other ZCL foci in Iran1111. Mirzaei A, Rouhani S, Kazerooni PA, Farahmand M, Parvizi P. Molecular detection and conventional identification of Leishmania species in reservoir hosts of zoonotic cutaneous leishmaniasis in Fars province, South of Iran. Iran J Parasitol 2013; 8:280-288.,1515. Mirzaei A, Rouhani S, Taherkhani H, Farahmand M, Kazemi B, Hedayati M, et al. Isolation and detection of Leishmania species among naturally infected Rhombomys opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkemen Sahara, North East of Iran. Exp Parasitol 2011; 129:375-380.,2525. Parvizi P, Moradi G, Akbari G, Ready PD, Farahmand M, Piazak N, et al. PCR Detection and sequencing of parasite ITS rDNA gene from reservoirs host of zoonotic cutaneous leishmaniasis in central of Iran. Parasitol Res 2008; 103:1273-1278.,3131. Mehrabani D, Motazedian MH, Hatam GR, Asgari Q, Owji SM, Oryan A. Leishmania Major in Tatera indica in Fasa, Southern Iran, microscopy, culture, isoenzyme, PCR and morphologic study. Asian J Anim Vet Adv 2011; 6:255-264.. This is the first report of L. major in only a small area of ZCL focus in Abarkouh. In our current publication, we also found L. major in P. papatasi, a proven vector of ZCL in Iran, in the same area in Abarkouh where L. major was isolated in rodents77. Parvizi P, Ready PD. Nested PCRs and sequencing of nuclear ITS rDNA fragments detect three Leishmania species of gerbils in sandflies from Iranian foci of zoonotic cutaneous leishmaniasis. TM & IH 2008; 13:1159-1171.,1010. Jafari R, Najafzadeh N, Sedaghat MM, Parvizi P. Molecular characterization of sandflies and Leishmania detection in main vector of zoonotic cutaneous leishmaniasis in Abarkouh district of Yazd province, Iran. Asian Pac J Trop Med 2013; 6:792-797.,3232. Killick-Kendrick R. Phlebotomine vectors of the leishmaniasis: A review. Med Vet Entomol 1990; 4:1-24..

Finding additional Leishmania infections in different rodent species compared with only one sandfly species can be explained by the fact that among sandflies, we mainly examined P. papatasi, and only a small number of other sandfly species were tested and found to be Leishmania negative, which did not provide sufficient for a precise result1010. Jafari R, Najafzadeh N, Sedaghat MM, Parvizi P. Molecular characterization of sandflies and Leishmania detection in main vector of zoonotic cutaneous leishmaniasis in Abarkouh district of Yazd province, Iran. Asian Pac J Trop Med 2013; 6:792-797.. In addition, only P. papatasi is able to develop L. major in its midgut and transfer the parasite to salivary glands to cause ZCL3333. Volf P, Benková I, Myšková J, Sádlová J, Campino L, Ravel C. Increased transmission potential of Leishmania major/Leishmania infantum hybrids. Int J Parasitol 2007; 37:589-593.. However, we analyzed all the captured rodent samples from different species, and therefore, we were able to identify Leishmania infections in at least three rodent species. The Leishmania infection rate in rodents as the reservoir host of ZCL is much higher than in sandflies as vectors, and in some cases, more than 50% of the samples were found to be infected with Leishmaniaparasites1414. Akhavan AA, Yaghoobi-Ershadi MR, Mirhendi H, Alimohammadian MH, Rassi Y, Shareghi N, Jafari R, Arandian MH, Abdoli H, Ghanei M, Jalali-zand N, Khamesipour A. Molecular epizootiology of Rodent leishmaniasis in hyperendemic area of Iran. Iran J Public Health 2010; 39:1-7..

Based on our experience in different ZCL foci in Iran, we expected to identify more Leishmania infections in reservoir hosts in Abarkouh district and to observe at least a small amount of variation in the ITS-rDNA gene of L. major in rodents. However, only one haplotype was found, and approximately 16% (11/68) of the tested rodents were infected with Leishmania parasites1111. Mirzaei A, Rouhani S, Kazerooni PA, Farahmand M, Parvizi P. Molecular detection and conventional identification of Leishmania species in reservoir hosts of zoonotic cutaneous leishmaniasis in Fars province, South of Iran. Iran J Parasitol 2013; 8:280-288.,1515. Mirzaei A, Rouhani S, Taherkhani H, Farahmand M, Kazemi B, Hedayati M, et al. Isolation and detection of Leishmania species among naturally infected Rhombomys opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkemen Sahara, North East of Iran. Exp Parasitol 2011; 129:375-380.,2525. Parvizi P, Moradi G, Akbari G, Ready PD, Farahmand M, Piazak N, et al. PCR Detection and sequencing of parasite ITS rDNA gene from reservoirs host of zoonotic cutaneous leishmaniasis in central of Iran. Parasitol Res 2008; 103:1273-1278.. After sequencing, only one haplotype of L. major, which is also the common haplotype present in Iran, including Fars and Isfahan Provinces, was detected from Abarkouh rodent samples (GenBank accession No. EF413075).

The low density of sampled rodents as well as Leishmaniaparasites may be due to a control program of the health care authorities of Abarkouh district that uses zinc phosphate poison and the destruction of rodents' barrowers to control ZCL.

According to reports of different ZCL foci in Iran, many haplotypes of L. major have been identified in sandflies, rodents and humans77. Parvizi P, Ready PD. Nested PCRs and sequencing of nuclear ITS rDNA fragments detect three Leishmania species of gerbils in sandflies from Iranian foci of zoonotic cutaneous leishmaniasis. TM & IH 2008; 13:1159-1171.,1111. Mirzaei A, Rouhani S, Kazerooni PA, Farahmand M, Parvizi P. Molecular detection and conventional identification of Leishmania species in reservoir hosts of zoonotic cutaneous leishmaniasis in Fars province, South of Iran. Iran J Parasitol 2013; 8:280-288.,1515. Mirzaei A, Rouhani S, Taherkhani H, Farahmand M, Kazemi B, Hedayati M, et al. Isolation and detection of Leishmania species among naturally infected Rhombomys opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkemen Sahara, North East of Iran. Exp Parasitol 2011; 129:375-380.,2727. Bordbar A, Parvizi P. High infection frequency, low diversity of Leishmania major and first detection of Leishmania turanica in human in northern Iran. Acta Trop 2014; 133:69-72.. The objective of the present study was to use molecular methods and different genes to identify additional Leishmania infections and various haplotypes; to this end, four different genes were employed to detect Leishmania infections in rodents and/or the numbers of haplotypes circulating in the area, but this method relies on a few sequences from all of the genes from our samples, and no variations were identified. We also used routine laboratory (conventional) methods, such as NNN cultures, microscopic observation and Balb/C mouse injection. Because most captured rodents died before being transferred to our lab, only a few live rodents were used for the conventional methods, and the infection rate was low. Only 2 infected samples were found by microscopic observation of the presence of amastigotes on slides and the appearance of a lesion after Balb/C mouse injection. Because the NNN cultures were prepared in the field and due to fungal infection in some cultures, no growth was shown in any of the cultures.

We employed five different genes during this investigation; the rodent Cyt b gene was amplified for accurate determination of the rodent's genus and species. For Leishmania infection, two mitochondrial genes (kDNA and Leishmania Cyt b) and two nuclear genes (ITS-rDNA and microsatellite ITS-rDNA) were used. In this investigation, the highest infection rate among rodents (8/68) was detected using the microsatellite ITS-rDNA gene because of its short tandemly repeated DNA sequence fragments and because it is highly specific. A comparison of the rest of the genes demonstrated that Cyt b as a mitochondrial gene is more sensitive for Leishmania detection (4/68) because of its high copy numbers per cell; however, nuclear genes are more specific, and of those, the ITS-rDNA gene (3/68), because it is homogenous and highly conserved with few intracellular polymorphisms, a linear genome and has readable sequences, is considered a suitable gene for sequencing, genus, species, strain and/or even haplotype detection.

Our Leishmania infection data in rodents are similar to the results of a parallel study among sandflies and suspected patients that was carried out near the time of our investigation1010. Jafari R, Najafzadeh N, Sedaghat MM, Parvizi P. Molecular characterization of sandflies and Leishmania detection in main vector of zoonotic cutaneous leishmaniasis in Abarkouh district of Yazd province, Iran. Asian Pac J Trop Med 2013; 6:792-797. (Parvizi P et al: unpublished data).

In previous investigations, reservoir hosts of ZCL have been distributed in different regions. R. opimus and M. libycus are dominant in the northeastern and central regions; M. libycus and T. indica in the central and southwestern regions and T. indica in southwestern and southern Iran1111. Mirzaei A, Rouhani S, Kazerooni PA, Farahmand M, Parvizi P. Molecular detection and conventional identification of Leishmania species in reservoir hosts of zoonotic cutaneous leishmaniasis in Fars province, South of Iran. Iran J Parasitol 2013; 8:280-288.,3434. Rouhani S, Mirzaei A, Spotin A, Parviz P. Novel identification of Leishmania major in Hemiechinus auritus and molecular detection of this parasite in Meriones libycus from an important foci of zoonotic cutaneous leishmaniasis in Iran. J Infect Public Health 2014; 7:210-217.. R. opimus and M. libycushave previously been found to be infected with L. major parasites from Golestan and Isfahan Provinces1111. Mirzaei A, Rouhani S, Kazerooni PA, Farahmand M, Parvizi P. Molecular detection and conventional identification of Leishmania species in reservoir hosts of zoonotic cutaneous leishmaniasis in Fars province, South of Iran. Iran J Parasitol 2013; 8:280-288.,1414. Akhavan AA, Yaghoobi-Ershadi MR, Mirhendi H, Alimohammadian MH, Rassi Y, Shareghi N, Jafari R, Arandian MH, Abdoli H, Ghanei M, Jalali-zand N, Khamesipour A. Molecular epizootiology of Rodent leishmaniasis in hyperendemic area of Iran. Iran J Public Health 2010; 39:1-7.,1515. Mirzaei A, Rouhani S, Taherkhani H, Farahmand M, Kazemi B, Hedayati M, et al. Isolation and detection of Leishmania species among naturally infected Rhombomys opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkemen Sahara, North East of Iran. Exp Parasitol 2011; 129:375-380.,2525. Parvizi P, Moradi G, Akbari G, Ready PD, Farahmand M, Piazak N, et al. PCR Detection and sequencing of parasite ITS rDNA gene from reservoirs host of zoonotic cutaneous leishmaniasis in central of Iran. Parasitol Res 2008; 103:1273-1278.. In addition, T. indica was found to be infected with L. major in Fars Province, Iran3131. Mehrabani D, Motazedian MH, Hatam GR, Asgari Q, Owji SM, Oryan A. Leishmania Major in Tatera indica in Fasa, Southern Iran, microscopy, culture, isoenzyme, PCR and morphologic study. Asian J Anim Vet Adv 2011; 6:255-264.. Interestingly, we were able to identify L. major infections in all three of these rodents within Abarkouh district of Yazd Province in central Iran.

In this survey, T. indica was captured for the first time in Abarkouh district; the existence of this rodent in the area may be explained by the fact that Abarkouh neighbors Fars Province, which is a known habitat for T. indica3131. Mehrabani D, Motazedian MH, Hatam GR, Asgari Q, Owji SM, Oryan A. Leishmania Major in Tatera indica in Fasa, Southern Iran, microscopy, culture, isoenzyme, PCR and morphologic study. Asian J Anim Vet Adv 2011; 6:255-264., and the rodents can be transported and/or migrate to Abarkouh from Fars and vice versa. The simultaneous existence of T. indica along with R. opimus and M. libycus as main and potential reservoir hosts of ZCL in Abarkouh district and the fact that Abarkouh has been largely neglected as an important ZCL focus gives this district an important role in the ZCL life cycle, epidemiology, prognosis and disease-control programs.

Leishmania major was firmly identified in R. opimus, M. libycus and T. indica, which indicates that at least these three rodent species can be incriminated as reservoir hosts of ZCL in this location. R. opimus was abundant and had a greater rate of L. major infection and should be incriminated as the main reservoir host of ZCL3535. Killick-Kendrick R, Ward RD. Ecology of the Leishmania. Parasitol 1981; 82:143-152..

CONFLICT OF INTEREST

The authors declare that there is no confl ict of interest.

FINANCIAL SUPPORT

This study was supported by the Pasteur Institute of Iran grant 501, awarded to Dr. Parviz Parvizi.

ACKNOWLEDGMENTS

The authors would like to thank the authorities Abarkouh Health Care for their help and support during this investigation. We thank Mehdi Baghban for helping with the field work and Elnaz AlaeeNovin for assistance in the Molecular Systematic Laboratory. A part of this research was funded through MSc studentships to Alireza Zamani based at the Pasteur Institute of Iran, Tehran, and registered for Islamic Azad University, Science and Research Branch of Fars, Iran.

REFERENCES

  • 1
    World Health Organization Control of Leishmaniasis. Technical report series 949 of WHO Expert Committee. Geneva: WHO; 2010.
  • 2
    Zare S, Baghestani S. Cutaneous Leishmaniasis in Hormozgan, Iran. Int J Dermatol 2001; 40:29-631.
  • 3
    Maraghi S, Samarbaf Zadeh A, Sarlak AA, Ghasemian M, Vazirianzadeh B. Identification of Cutaneous Leishmaniasis Agents by Nested Polymerase Chain Reaction (Nested-PCR) in Shush City, Khuzestan Province, Iran. Iran J Parasitol 2007; 2:13-15.
  • 4
    Jahani MR, Gharavi MJ, Hadi Shirzad H. Passive Detection of Cutaneous Leishmaniasis in Police Personnel Deployed in the Provinces of Isfahan, Ilam, Bushehr, Khorasan and Khuzestan, Iran. Iran J Public Health 2003; 32:23-27.
  • 5
    Sharbatkhori M, Spotin A, Taherkhani H, Roshanghalb M, Parvizi P. Molecular variation in Leishmania parasites from sandflies species of a zoonotic cutaneous leishmaniasis in northeast of Iran. J Vector Borne Dis 2013; 51:16-21.
  • 6
    Alavinia SM, Arzamani K, Reihani MH, Jafari J. Some epidemiological aspects of cutaneous leishmaniasis in Northern Khorasan Province, Iran. Iran J Arthropod Borne Dis 2009; 3:50-54.
  • 7
    Parvizi P, Ready PD. Nested PCRs and sequencing of nuclear ITS rDNA fragments detect three Leishmania species of gerbils in sandflies from Iranian foci of zoonotic cutaneous leishmaniasis. TM & IH 2008; 13:1159-1171.
  • 8
    Ready PD. Biology of Phlebotomine sand flies as vectors of disease agents. Ann Rev Entomol 2013; 58:227-250.
  • 9
    Parvizi P, Bordbar A, Najafzadeh N. Detection of Wolbachia pipientis, including a new strain containing the wsp gene, in two sister species of Paraphlebotomus sandflies, potential vectors of zoonotic cutaneous leishmaniasis. Mem Inst Oswaldo Cruz 2013; 108:414-420.
  • 10
    Jafari R, Najafzadeh N, Sedaghat MM, Parvizi P. Molecular characterization of sandflies and Leishmania detection in main vector of zoonotic cutaneous leishmaniasis in Abarkouh district of Yazd province, Iran. Asian Pac J Trop Med 2013; 6:792-797.
  • 11
    Mirzaei A, Rouhani S, Kazerooni PA, Farahmand M, Parvizi P. Molecular detection and conventional identification of Leishmania species in reservoir hosts of zoonotic cutaneous leishmaniasis in Fars province, South of Iran. Iran J Parasitol 2013; 8:280-288.
  • 12
    Ready PD. Leishmaniasis emergence and climate change. Rev Sci Tech 2008; 27:399-412.
  • 13
    Safari Z, Saadati M, Doroudian M, Hossaini SM, Hashemi M, Rezai zarchi S. Cutaneous leishmaniasis characterization in the Center Part of Iran; is that an emerging disease? Adv Environ Biol 2011; 5:3464-3470.
  • 14
    Akhavan AA, Yaghoobi-Ershadi MR, Mirhendi H, Alimohammadian MH, Rassi Y, Shareghi N, Jafari R, Arandian MH, Abdoli H, Ghanei M, Jalali-zand N, Khamesipour A. Molecular epizootiology of Rodent leishmaniasis in hyperendemic area of Iran. Iran J Public Health 2010; 39:1-7.
  • 15
    Mirzaei A, Rouhani S, Taherkhani H, Farahmand M, Kazemi B, Hedayati M, et al. Isolation and detection of Leishmania species among naturally infected Rhombomys opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkemen Sahara, North East of Iran. Exp Parasitol 2011; 129:375-380.
  • 16
    Parvizi P, Mauricio I, Aransay AM, Miles MA, Ready PD. First detection of Leishmania major in peridomestic Iranian sandflies: comparison of nested PCR of nuclear ITS ribosomal DNA and semi-nested PCR of minicircle kinetoplast DNA. Acta Trop 2005; 93:75-83.
  • 17
    Luyo-Acero GE, Uezato H, Oshiro M, Takei K, Kariya K, Katakura K, et al. Sequence variation of the Cytochrome b gene of various human infecting members of the genus Leishmania and their phylogeny. Parasitol 2004; 128:483-491.
  • 18
    Maleki-Ravasan N, Oshaghi MA, Javadian E, Rassi Y, Sadraei J, Mohtarami F. Blood meal identification in field-captured sandflies: comparison of PCR RFLP and ELISA assays. Iran J Arthropod Borne Dis 2009; 3:8-18.
  • 19
    Oryan A, Shirian S, Tabandeh MR, Hatam GR, Randau G, Daneshbod Y. Genetic diversity of Leishmania major strains isolated from different clinical forms of cutaneous leishmaniasis in southern Iran based on minicircle kDNA. Infect Genet Evol 2013; 19:226-31.
  • 20
    Jamjoom MB, Ashford RW, Bates PA, Kemp SJ, Noyes HA. Polymorphic microsatellite repeats are not conserved between Leishmania donovani and Leishmania major. Mol Ecol Notes 2002; 2:104-106.
  • 21
    Kuhls K, Chicharro C, Canavate C, Cortes S, Campino L, Haralambous C, et al. Differentiation, Gene Flow, among European populations of Leishmania infantum MON-1. PLoS Negl Trop Dis 2008; 2:261.
  • 22
    Russell R, Iribar MP, Lambson B, Brewster S, Blackwell JM, Dye C, et al. Intra and inter-specific microsatellite variation in the Leishmania subgenus Viannia. Mol Biochem Parasit 1999; 103:71-77.
  • 23
    Degli esposti M, De Vries S, Crimi M, Ghelly A, Patarnello T, Meyer A. Mitochondrial cytochrome b: evolution and structure of the protein. Biochim Biophys Acta 1993; 1143:243-271.
  • 24
    Ziaei H. A field guide for identifying of Iranian desert mammalians. 1st ed. Tehran: Iranian Environment Organization; 1996.
  • 25
    Parvizi P, Moradi G, Akbari G, Ready PD, Farahmand M, Piazak N, et al. PCR Detection and sequencing of parasite ITS rDNA gene from reservoirs host of zoonotic cutaneous leishmaniasis in central of Iran. Parasitol Res 2008; 103:1273-1278.
  • 26
    Kent R, Norris DE. Identification of mammalian blood meals in mosquitoes by a multiplex polymerase chain reaction targeting cytochrome b. Am J Trop Med Hyg 2005; 73:336-342.
  • 27
    Bordbar A, Parvizi P. High infection frequency, low diversity of Leishmania major and first detection of Leishmania turanica in human in northern Iran. Acta Trop 2014; 133:69-72.
  • 28
    Brewster S, Barker DC. Analysis of minicircle classes in Leishmania (Viannia) species. T Roy Soc Trop Med H 2002; 96 (suppl I):55-63.
  • 29
    Parvizi P, Alaeenovin E, Kazerooni PA, Ready PD. Low diversity of Leishmania parasites in sandflies and the absence of the great gerbil in foci of zoonotic cutaneous leishmaniasisin Fars province, southern Iran. T Roy Soc Trop Med H 2013; 107:356-362.
  • 30
    Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, et al. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011; 10:2731-2739.
  • 31
    Mehrabani D, Motazedian MH, Hatam GR, Asgari Q, Owji SM, Oryan A. Leishmania Major in Tatera indica in Fasa, Southern Iran, microscopy, culture, isoenzyme, PCR and morphologic study. Asian J Anim Vet Adv 2011; 6:255-264.
  • 32
    Killick-Kendrick R. Phlebotomine vectors of the leishmaniasis: A review. Med Vet Entomol 1990; 4:1-24.
  • 33
    Volf P, Benková I, Myšková J, Sádlová J, Campino L, Ravel C. Increased transmission potential of Leishmania major/Leishmania infantum hybrids. Int J Parasitol 2007; 37:589-593.
  • 34
    Rouhani S, Mirzaei A, Spotin A, Parviz P. Novel identification of Leishmania major in Hemiechinus auritus and molecular detection of this parasite in Meriones libycus from an important foci of zoonotic cutaneous leishmaniasis in Iran. J Infect Public Health 2014; 7:210-217.
  • 35
    Killick-Kendrick R, Ward RD. Ecology of the Leishmania. Parasitol 1981; 82:143-152.

Publication Dates

  • Publication in this collection
    Oct 2014

History

  • Received
    9 Sept 2014
  • Accepted
    4 Oct 2014
Sociedade Brasileira de Medicina Tropical - SBMT Caixa Postal 118, 38001-970 Uberaba MG Brazil, Tel.: +55 34 3318-5255 / +55 34 3318-5636/ +55 34 3318-5287, http://rsbmt.org.br/ - Uberaba - MG - Brazil
E-mail: rsbmt@uftm.edu.br